Fibrin is a white insoluble fibrous protein formed from fibrinogen by the action of thrombin. In the clotting of blood, fibrin forms the structural scaffold of a thrombus, which is a clot of blood formed within a blood vessel that remains attached to its place of origin. Under normal conditions the blood clotting system is maintained in equilibrium and the fibrin deposits are dissolved by the fibrinolytic enzyme system. Unfortunately, events such as vascular damage, activation/stimulation of platelets, and activation of the coagulation cascade may disturb the equilibrium, which can result in thrombosis or the blockage of a blood vessel by a blood clot.
Intravascular thrombosis is one of the most frequent pathological events accounting for greater than 50% of all deaths as well as a variety of other serious clinical problems. Most spontaneously developing vascular obstructions are due to the formation of intravascular blood clots, also known as thrombi. Small fragments of a clot may detach from the body of the clot and travel through the circulatory system to lodge in distant organs and initiate further clot formation. Myocardial infarction, occlusive stroke, deep venous thrombosis (DVT) and peripheral arterial disease are well-known consequences of thromboembolic phenomena.
Plasminogen activators are currently the favored agents employed in thrombolytic therapy, all of which convert plasminogen to plasmin and promote fibrinolysis by disrupting the fibrin matrix (M. A. Creager and V. J. Dzau, Vascular Diseases of the Extremities, ppgs. 1398-1406 in Harrison's Principles of Internal Medicine, 14th ed., Fauci et al, editors, McGraw-Hill Co., New York, 1998; the contents of which is incorporated herein by reference in its entirety).
The most widely used plasminogen activators include a recombinant form of tissue-type plasminogen activator (tPA), urokinase (UK) and streptokinase (SK), as well as a new generation of plasminogen activators selected for improved pharmacokinetics and fibrin-binding properties. All of these plasminogen activators, however, by virtue of their mechanism of action, act indirectly and require an adequate supply of their common substrate, plasminogen, at the site of the thrombus to effect lysis.
UK and tPA convert plasminogen to plasmin directly by cleaving the Arg560-Val561 peptide bond. The resulting two polypeptide chains of plasmin are held together by two interchain disulfide bridges. The light chain of 25 kDa carries the catalytic center and is homologous to trypsin and other serine proteases. The heavy chain (60 kDa) consists of five triple-loop kringle structures with highly similar amino acid sequences. Some of these kringles contain so-called lysine-binding sites that are responsible for plasminogen and plasmin interaction with fibrin, α2-antiplasmin or other proteins. SK and staphylokinase activate plasminogen indirectly by forming a complex with plasminogen, which subsequently behaves as a plasminogen activator to activate other plasminogen molecules by cleaving the arginyl-valine bond.
Although thrombolytic drugs, such as tissue plasminogen activator (tPA), streptokinase, and urokinase, have been successfully employed clinically to reduce the extent of a thrombotic occlusion of a blood vessel, it appears that serious limitations persist with regard to their use in current thrombolytic therapy. For example, because the activation of plasminogen by tPA is fibrin dependent for full proteolytic activity to be realized (Haber et al. 1989), excessive bleeding may result as a side effect of its use. Other adverse sequelae associated with the use of these thrombolytic agents include myocardial infarction, occlusive stroke, deep venous thrombosis and peripheral arterial disease.
Additionally, the known plasminogen activators currently used suffer from several limitations that impact their overall usefulness in the elimination of a thrombus. For example, at best, the use of current thrombolytic therapy results in restored vascular blood flow within 90 min in approximately 50% of patients, while acute coronary re-occlusion occurs in roughly 10% of patients. Coronary recanalization requires on average 45 minutes or more, and intracerebral hemorrhage occurs in 0.3% to 0.7% of patients. Residual mortality is at least 50% of the mortality level in the absence of thrombolysis treatment.
A different approach to avoid the problems associated with the systemic administration of a plasminogen activator to generate sufficient plasmin at the site of the thrombus, is to directly administer the plasmin itself to the patient.
In U.S. Pat. No. 5,288,489, Reich et al., disclose a fibrinolytic treatment that includes parenterally introducing plasmin into the body of a patient. The concentration and time of treatment were selected to be sufficient to allow adequate active plasmin to attain a concentration at the site of an intravascular thrombus that is sufficient to lyse the thrombus or to reduce circulating fibrinogen levels. However, the necessity of generating the plasmin from plasminogen immediately prior to its introduction into the body is also disclosed.
In contrast, U.S. Pat. No. 3,950,513 to Jenson teaches that plasmin compositions may be stabilized at pH 7.0 by including a physiological non-toxic amino acid. This method dilutes stock plasmin solutions stored at low pH with the neutralizing amino acid immediately prior to administration. There are advantages, however, in maintaining low pH of the plasmin composition as long as possible to minimize autodegradation. Ideally, the plasmin will be retained at a low pH until encountering the target fibrin.
Yago et al. disclose plasmin compositions useful as a diagnostic reagent in U.S. Pat. No. 5,879,923. The compositions of Yago et al. comprise plasmin and an additional component which may be 1) an oligopeptide comprising at least two amino acids, or 2) at least two amino acids, or 3) a single amino acid and a polyhydric alcohol. However, the compositions of Yago et al. are formulated at a neutral pH to maintain the enzymatic activity of plasmin.
Plasmin as a potential thrombolytic agent has numerous technical difficulties. These difficulties include the challenge of preparing pure plasmin that is free of all functional traces of the plasminogem activator used to convert plasmin from its inactive precursor, plasminogen. Preparations of plasmin are typically extensively contaminated by plasminogen activator, streptokinase or urokinase and the thrombolytic activity was, therefore, attributed to the contaminating plasminogen activators rather than to plasmin itself. The contaminating plasminogen activators could also trigger systemic bleeding other than at the targeted site of thrombosis. A drawback of streptokinase containing plasmin preparations is that streptokinase can cause adverse immune reactions including fever and anaphylactic shock.
One of the more important technical factors limiting clinical use of plasmin is that plasmin, as a serine protease with broad specificity, is highly prone to autodegradation and loss of activity. This circumstance provides severe challenges to the production of high-quality plasmin, to the stable formulation of this active protease for prolonged periods of storage prior to use, and to safe and effective administration of plasmin to human patients suffering from occlusive thrombi. Thus, there is need for a method of producing stable plasmin.